Mitochondria are dynamic organelles playing essential metabolic and signaling functions in cells. Their ultrastructure has been largely investigated with electron microscopy (EM) techniques. Super-resolution microscopy approaches such as direct stochastic optical reconstruction microscopy (dSTORM) provide a fluorescent-based, quantitative alternative to EM. However, dSTORM is mainly used to image integral mitochondrial proteins, and there is little or no information on proteins transiently present at this compartment. Here, we first benchmark the power of dSTORM to resolve protein proximities on individual mitochondrial subcompartments, coupled to Geo-coPositioning System (GcoPS) to quantify the degree of protein colocalization. With our dSTORM/GcoPS method, we then analyze the submitochondrial distribution of the cancer-related Aurora kinase A/AURKA, a protein localized at various subcellular locations including mitochondria. We show that dSTORM provides sufficient spatial resolution to detect a large pool of endogenous AURKA within the matrix, and we also uncover a second pool of the kinase at the Outer Mitochondrial Membrane (OMM). We conclude by demonstrating that an aldehyde-based fixation allows for a more specific detection of the OMM pool of AURKA. Our results indicate that dSTORM coupled to GcoPS colocalization analysis is a suitable approach to explore the compartmentalization of non-integral mitochondrial proteins as AURKA, in a qualitative and quantitative manner. This method also opens up the possibility of analyzing the proximity between AURKA and its multiple mitochondrial partners with exquisite spatial resolution, thereby allowing novel insights into the mitochondrial functions controlled by AURKA.

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